Biological thiols such as Cysteine (Cys), Homocysteine (Hcy) and Glutathione (GSH) play crucial roles in maintaining cellular antioxidant defence system. Among them cysteine plays many important roles in living systems. Cysteine is one of the three main precursors required for GSH synthesis. The deficiency of this compound causes many diseases such as slowed growth in children, depigmentation of hair, edema, liver damage, skin lesions, and weakness. An elevated level of Hcy is a risk factor for cardio-vascular disease, dementia and Alzheimer's disease. Abnormal levels of GSH is connected to many diseases such as HIV, cell death and aging. Thus, detection and discrimination of these thiol containing molecules are of great importance. Because of the similar structure and reactivity, distinction among biothiols is a challenging task. The respective concentration level of Cys in human plasma is typically 240-360 μM. Estimation of these amino thiols in human blood plasma is essential for understanding the role of these in the pathogenesis of vascular diseases.

The past two decade has seen significant effort being devoted to the development of optical probes for the selective recognition of thiol containing amino acids. There are many strategies for sensing biothiols, based on Micheal addition, cyclization with aldehydes, disulfide cleavage and others.
Chinese Pat. No. 103570701 discloses a coumarin derivative and preparation method and application, the coumarin derivatives, the Formula C13H6N2O4. Preparation of the present invention, 2,2-dicyano-3-(7-hydroxy-4-coumarin-yl) oxirane as a living body to selectively detect cysteine fluorescent probe, can also be used for fluorescence cysteine labeled cells within, for the diagnosis of related diseases in clinical medicine to help.
Chinese Pat. No. 103788076 discloses a method for quantitative detection of cysteine, is based on N-[4-methylcoumarin-7-yl] maleimide in HEPES pH 7.4 solution quantitatively detect cysteine acid content. The detection method for cysteine showed high sensitivity and selectivity, the detection process is simple, sensitive, rapid, accurate test results.
Article titled “7-Diethylamino-3-{(E)-4-[(E)-2-(pyridin-4-yl)ethenyl]styryl}-2H-chromen-2-one” by Li-Ping Zhou et al. published in Acta Crystallographica Section E Structure Reports Online, 2014, 70(2), o176 reports coumarin derivative, C28H26N2O2, the coumarin unit is approximately planar, with a maximum deviation of 0.048 (3) Å. The central benzene ring is oriented at dihedral angles of 30.15 (14) and 10.51 (11°), respectively, to the pyridine ring and coumarin ring system. In the crystal, weak C—H . . . O and C—H . . . N hydrogen bonds and weak C—H . . . π interactions link the molecules into a three-dimensional supramolecular architecture.
Article titled “Thiol-chromene click chemistry: a coumarin-based derivative and its use as regenerable thiol probe and in bioimaging applications” by Yang Y et al. published in Biosensors and Bioelectronics 2013; 47:pp 300-6 reports the synthesis and characterization of a coumarin-chromene (8, 9-dihydro-2H-cyclopenta[b]pyrano[2,3-f]chromene-2,10(7aH)-dione) (1) derivative and its use for thiol chemosensing in water was reported. Experimental details showed 1 acts as a probe for the detection of thiols including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), whereas amino acids which do not contain thiols induced no changes in UV-vis spectra and fluorescence emission properties of 1. A possible detection mechanism is a nucleophilic attack of thiols to the α,β-unsaturated ketone in 1 that resulted in a fluorescent coumarin derivative. Further studies showed that 1-thiol derivatives can be applied to the design of regenerative chemodosimeters for Cu(2+), Hg(2+) and Cd(2+) in water based on M(n+)-promoted desulfurization and recovery of 1. Furthermore, the optical properties of the probe and its Cys-addition product were theoretically studied. The ability of probe 1 to detect thiols in living cells (HepG2 cells) via an enhancement of the fluorescence was proved. Moreover, the applicability of 1 for the direct determination of biorelevant thiols in a complex matrix such as human plasma was also demonstrated.
Article titled “Nitroolefin-based coumarin as a colorimetric and fluorescent dual probe for biothiols” by Sun Y Q et al. published in Chemical Communications, 2011; 47(39) pp 11029-31 reports a coumarin-based thiol probe featuring the 1,4-addition reaction of thiols to nitroolefin was reported. The molecular probe exhibited higher selectivity toward biothiols (Cys, Hcy and GSH) than other amino acids.
Article titled “Coumarin-Derived Fluorescent Chemosensors.” by Raman G et al. published in Phytochem Analysis, 2005; 16(3):155-60 reports Coumarin-derived fluorescent chemosensors for thiols. Study on fluorescent and colorimetric probes for detection of thiols has received much attention and many coumarin-derived fluorescent chemosensors for detection of thiols have been reported.
Article titled “Colorimetric detection of thiols using a chromene molecule” by Fang-Jun Huo et al. published in Organic Letters, 2009, 11(21), pp 4918-4921 reports a new thiol-containing colorimetric probe has been developed by using a chromene derivative, 7-nitro-2,3-dihydro-1H-cyclopenta[b]chromen-1-one. The molecule exhibited high selectivity and sensitivity for detecting thiol species as cysteine, homocysteine, and glutathione in aqueous solution through a rapid visual color change from colorless to yellow. The pH range (5.0-10.0) for determination of cysteine was also studied and the result showed that the system had the above-mentioned UV/visible light absorption with no significant difference within the pH range 7.0-10.0. Therefore selected the physiological condition of a HEPES 10 mM, pH 7.0 buffer aqueous solution. Systems with pH<7.0 will not exhibit a notable change in either color or UV/visible spectrum.
Article titled “The development of a fluorescence turn-on sensor for cysteine, glutathione and other biothiols. A kinetic study” by Olimpo Garcia-Beltran et al. published in Tetrahedron Letters, 2011, 52, pp 6606-6609 reports two fluorescence probes for the detection of cysteine (Cys), glutathione (GSH) and other biothiols, such as homocysteine (Hcy) and cysteinyl-glycine (Cys-Gly), were developed. These molecular probes are coumarin-based derivatives containing a chalcone-like moiety that reacts with biothiols through a Michael addition reaction, leading to strong fluorescence enhancements. The reactivity of the tested biothiols toward both probes (ChC1 and ChC2) follows the order Cys>GSH>Hcy>Cys-Gly, ChC1 being less reactive than ChC2. Possible interference with other amino acids was assessed.
ChC1 and ChC2 display a highly selective fluorescence enhancement with thiols, allowing these probes to be used for fluorimetric thiol determination in SH-SY5Y cells.
Article titled “Synthesis of coumarin derivatives as fluorescent probes for membrane and cell dynamics studies” by Olimpo Garcia-Beltran et al. published in European Journal of Medicinal Chemistry, 2014, 76, pp 79-86 reports three coumarin-derived fluorescent probes, 3-acetyl-7-[(6-bromohexyl)oxy]-2H-chromen-2-one (FM1), 7-[(6-bromohexyl)oxy]-4-methyl-2H-chromen-2-one (FM2) and ethyl 2-{7-[(6-bromohexyl)oxy]-2-oxo-2H-chromen-4-yl}acetate (FM3), are described, with their photophysical constants. All absorption and emission spectra were measured in a mixture of ACN:aqueous 20 mM HEPES buffer, pH 7.4, 1.1.
Article titled “New chemodosimetric reagents as ratiometric probes for cysteine and homocysteine and possible detection in living cells and in blood plasma” by Priyadip Das et al. published in Chemistry, 2012; 18(48), pp 15382-93 reports rationally designed and synthesized two new reagents (L1 and L2), each bearing a pendant aldehyde functionality. This aldehyde group can take part in cyclization reactions with b-org-amino thiols to yield the corresponding thiazolidine and thiazinane derivatives, respectively. The recognition of Cys/Hcy by L1 and L2 was investigated by monitoring the changes in the electronic spectra of these receptors upon the formation of their respective thiazolidine moieties. Thus, L1 and L2 were each treated with varying concentrations of Cys and Hcy in appropriate DMSO/aqueous HEPES buffer (pH 7.4) media. Upon addition of increasing amounts of Cys or Hcy to a solution of L1 in DMSO/aqueous HEPES buffer (50 mm, 3:2 (v/v), pH 7.4), the absorption band at around 397 nm was found to decrease gradually with a concomitant increase at around 460 nm. experiments were carried out to prove the above sensing mechanism. Firstly, the product mixture of the reaction of cysteamine (cysteamine was selected instead of Cys because of its good solubility in organic solvents) with 4 in CH3OH was separated and 1,4-thiazepan-5-one (6) and the parent SNF were obtained, respectively. The structure of 6 was identified by 1H NMR, 13C NMR and HRMS. The formation of SNF was confirmed by a major peak at m/z 395.0929, corresponding to [SNF—H]−(calc. 395.0919 for C25H15O5) was shown in the HRMS data. Secondly, cysteamine and 3-mercaptopropanoic acid (MPA) were introduced to the solution of 4, respectively, and it was observed that the former gives a prominent increase in fluorescence emission but the latter produces weak fluorescence increase at the same conditions. Lastly, N-acetyl-1-cysteine (NAC) was added to a solution of 4 and almost no fluorescence increase was observed under the same conditions. The above experiments serve as strong evidence that both sulfhydril and amino groups (the NAC amine is blocked) of Cys are responsible for the signal.
Article titled “A seminaphthofluorescein-based fluorescent chemodosimeter for the highly selective detection of cysteine” by Xiaofeng Yang et al. published in Organic and Molecular Chemistry, 2012, 10(14), pp 2739-2741 reports a fluorescent chemodosimeter for cysteine detection was developed based on a tandem conjugate addition and intramolecular cyclization reaction. The method exhibited an excellent selectivity for cysteine over other biothiols such as homocysteine and glutathione. Color changes of the solution of 4 (10 μM) in the presence of different biothiols (2 equiv.) in 1.0 mM CTAB media buffered at pH 7.4 (Hepes buffer, 20 mM) after 25 min.
Article titled “Dicyclohexylcarbodiimide (DCC)” by Miroslav Kvasnica published in Synlett, 2007(14), pp 2306-2307 reports synthesis of amides, lactams and peptides by using DCC and DMAP. Heterocyclization reactions: DCC is widely used as a reagent (or reactant) in the synthesis of heterocycles. Jeon and co-workers published the solid-phase synthesis of 2-amino-2-thiazolines using the DCC-mediated cyclization of N-(2-hydroxyethyl) thioureas.
Article titled “A cysteine-selective fluorescent probe for the cellular detection of cysteine” by Jung H S et al. published in Biomaterials, 2012, 33(3), pp 945-953 reports a series of coumarin fluorophores, each bearing a double bond conjugated quinoline unit that can undergo a Michael-type reaction with thiol-containing compounds, is reported. These systems, designed to provide so-called turn-on changes in fluorescence response when exposed to thiols, act as fluorescent chemical sensors for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH).
Article titled “Synthesis and vasorelaxant and platelet antiaggregatory activities of a new series of 6-Halo-3-phenylcoumarins” by Elias Quezada et al. published in Molecules 2010, 15, pp 270-279 reports 3-phenylcoumarins were prepared from the conveniently substituted phenylacetic acids, the appropriate salicylaldehyde and dicyclohexylcarbodiimide (DCC) by a Perkin reaction in dimethylsulfoxide (DMSO).
Article titled “The synthesis, characterization and properties of coumarin glycosides and coumarin-strapped calixpyrrole derivatives” by LiYang published in Master's thesis in 2011 reports designed and synthesized coumarin-strapped calix[4]pyrrole derivatives. With 2-Hydroxy-1-naphthaldehyde, 4-(Diethylamino) salicylaldehyde, salicylaldehyde and diethyl malonate as raw materials, pyridine and acetic acid as catalysts, coumarin esters were synthesized, and through hydrolysis reaction, coumarin acid compounds were got. Using DCC/DMAP method and carbonyl chlorides method, coumarin-Strapped calix pyrrole derivatives were synthesized.
Article titled “Synthesis of liquid crystals materials derived from oxadiazole, isoxazole and tetrazole heterocycles” by Daniela Rubia dos Santos et al. published in ARKIVOC 2008 (xvii) 157-166 reports The synthesis is described of new liquid crystalline heteroaromatic compounds containing the five-membered isoxazole, tetrazole and 1,2,4-oxadiazole rings.
Article titled “Cesium carbonate catalyzed efficient synthesis of quinazoline-2,4(1H,3H)-diones using carbon dioxide and 2-aminobenzonitriles” by Yogesh P. Patil et al. published in Green Chemistry Letters and Reviews, 2008, 1 (2) reports an efficient protocol for the synthesis of quinazoline-2,4(1H,3H)-diones derivatives from 2-aminobenzonitriles with carbon dioxide using catalytic amount of cesium carbonate has been developed.
Article titled “5-Membered N-heterocyclic compounds by dimethyl carbonate chemistry” by Fabio Arico et al. published in Green Chemistry, 2012, 14, 58 reports synthesis of N-methoxycarbonyl pyrrolidine starting from 4-amino-1-butanol using DMC as solvent and reagent in the presence of catalytic amount of base. Among the catalysts used, alkali carbonates and in particular Cs2CO3 resulted the more efficient ones (62% yield).
Article titled “Synthesis of esters derived from 2,3,4-tri-O-benzyl-α-D-methylglucoside” by Tiago A. D. Pinto et al. published in ARKIVOC 2012 (vi) 185-193 reports 2,3,4-Tri-O-benzyl-α-D-methylglucoside was prepared and reacted with several acids: benzoic, phenylacetic, 2-(3-bromo-propoxy)-benzoic, acetylsalicylic and 4-(toluene-4-sulfonylamino)-benzoic. The products were isolated with low to fair yields and fully characterized by usual analytical techniques.
Article titled “Synthesis, characterization and antioxidant activities of Schiff bases are of cholesterol” by Madasamy Kumar et al. published in Journal of Saudi Chemical Society, Available online 4 Apr. 2014 reports synthesis of cholesterylbromo alkyl derivatives and synthesis of cholesteryl aldehyde derivatives.
Article titled “Palladium-Catalysed C—C Coupling: Then and Now” by Chris Barnard published in Platinum Metals Rev., 2008, 52, (1), pp 38-45 reports Heck reactions.
Articled titled “The Synthesis of beta-Nitrostyrenes” by C. B. Gairaud et al. published in Journal of Organic Chemistry, 1953, 18, pp 1-3 reports the condensation of nitroalkanes with benzaldehydes to give β-nitrostyrenes has usually been catalyzed by bases such as alcoholic potassium hydroxide or alcoholic methylamine although the reaction was first carried out using an acid catalyst, zinc chloride.
Article titled “Nitrostyrene” by David E. Worrall published in Organic Syntheses, Coll. Vol. 1, p. 413 (1941); Vol. 9, p. 66 (1929) reports synthesis of nitrostyrene.
Article titled “Coumarin-derived fluorescent chemosensors” by Hongqi Li et al. published in Advances in Chemical Sensors reports coumarin-derived fluorescent chemosensors for H2O2, O2, hydroxyl radicals or chemical warfare agents in milk samples.
Article titled “Determination of lanthionine by thin-layer chromatography” by Mario Marzona et al. published in Journal of Chromatography A, 1968, 32, pp 755-757 reports determination of lanthionine by thin-layer chromatography.
However, development of probes for specific discrimination of biothiols is an unmet need in the art. Probes that are selective to any one of these amino biothiols are very rare in literature. Probes that give specific response with colour change as well as emission change are much needed. Especially those probes which allows real time monitoring without the aid of any instrumental techniques are highly recommended as for as the practical utility is concerned. There are no reports on detection of thiols using simple test strips. Most importantly, those probes which are capable of detecting Cysteine in protein residues are of much significance as such probes are desired for studying complex structures of proteins and their dynamics.
To fulfill this need for the selective detection of thiols, specifically biothiols, with a high degree of selectivity towards cysteine, the inventors have disclosed novel coumarin, and process of synthesis thereof.